Biofilms are populations of bacteria or fungi growing attached to an inert or living surface. Mounting evidence has shown that biofilms constitute a significant threat to human health. The Public Health Service estimates that biofilms are responsible for more than 80% of bacterial infections in humans (National Institutes of Health, 1998 RFA# DE-98-006). Examples of diseases caused by biofilms include dental caries, periodontitis, cystic fibrosis pneumonia, native valve endocarditis, and otitis media (Costerton et al. Science 1999 284:1318-1322), as well as infection of various medical devices such as urinary catheters, mechanical heart valves, cardiac pacemakers, prosthetic joints, and contact lenses (Donlan, R. M. 2001 Emerging Infect. Dis. 7:277-281). Fungi also form biofilms of clinical significance, for example Candida infections. Biofilm infections afflict tens of millions of patients in the U.S. annually and require a significant expenditure of health care dollars (Costerton et al. Science 1999 284:1318-1322). Bacteria growing in biofilms exhibit increased resistance to antimicrobial agents and are nearly impossible to eradicate. New methods for treating biofilm infections are needed.
Bacteria in a biofilm are enmeshed in an extracellular polysaccharide (EPS) substance that holds the bacteria together in a mass, and firmly attaches the bacterial mass to the underlying surface. Previous studies have demonstrated that enzymes that degrade EPS are capable of causing the detachment of cells from biofilms. For example, over expression of alginate lyase, an enzyme that catalyzes the degradation of the EPS alginate, causes colonies of Pseudomonas aeruginosa to become less adherent to surfaces (Boyd, A. and Chakrabarty, A. M. Appl. Environ. Microbiol. 1994 60:2355-2359). Alginate lyase has been suggested for use in treating P. aeruginosa infections in the lungs of cystic fibrosis patients (Mrsny et al. Pulm. Pharmacol. 1994 7:357-366). A similar polysaccharide lyase has been shown to be produced by P. fluorescens (Allison et al. FEMS Microbiol. Lett. 1998 167:179-184). Two other EPS-degrading enzymes, endo-β-1,4-mannanase from the plant pathogen Xanthomonas campestris (Dow et al. Proc. Nat. Acad. Sci. USA 2003 100:10995-11000) and disaggretase from the methanogenic archaebacterium Methanosarcina mazei (Liu et al. Appl. Environ. Microbiol. 1985 49:608-613), have also been shown to cause biofilm cell detachment. In the case of X. campestris, production of the EPS-degrading enzyme was required for full virulence of the bacteria in plants. Detachment of cells from biofilm colonies of the dental pathogen Streptococcus mutans was shown to be caused by an unidentified endogenous enzymatic activity (Lee et al. Infect. Immun. 1996 64:1035-1038). A complex mixture of polysaccharide-hydrolyzing enzymes was shown to remove biofilms from steel and polypropylene substrata (Johansen et al. Appl. Environ. Microbiol. 1997 63:3724-3728). These findings indicate that EPS-degrading enzymes can potentially be used as agents to remove. biofilms from surfaces.
Although enzymes are commonly used to remove biofilms in industrial environments, no studies have investigated the potential use of enzymes as agents for the removal of biofilms in clinical environments. Of particular concern in the clinic are biofilm infections of indwelling medical devices, especially intravascular catheters. Catheter infections are common in hospitalized patients and are associated with high levels of morbidity and mortality. A promising new approach to treating these infections is the use of catheters that are coated or impregnated with antimicrobial agents such as antibiotics (Schierholz et al. J. Antimicrobial. Chemother. 2000 46:45-50), silver (Bechert et al. Infection 1999 27:S24-S29), and peptide quorum-sensing inhibitors (Balaban et al. J. Infect. Dis. 2003 187:625-630). Numerous studies have demonstrated that medical devices with antimicrobial activity decrease the risk of bacterial colonization and infection (Tcholakian, R. K. and Raad, I. I. Antimicrob. Agents Chemother. 2001 45:1990-1993).
The present invention provides isolated proteins and active fragments and variants thereof and nucleic acid sequences encoding such proteins and active fragments and variants thereof involved in detachment of bacterial cells. Methods for modulating detachment of biofilm cells of bacteria or fungi and identifying agents which modulate bacterial or fungal detachment via these proteins and active fragments and variants thereof and/or nucleic acid sequences are also provided.